Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Bichel [Bichel, Nature 231: 449-450 (1971)] reported that removing most of the tumor from mice bearing ascites tumors at a plateau of tumor growth, was followed by a marked increase in the growth of the remaining tumor cells. Injection of cell-free ascites, obtained from mice bearing fully developed ascites tumors, into mice with growing ascites tumors, resulted in a pronounced inhibition of ascites growth. Bichel, supra, also observed that two surgically joined mice (parabiotic), one mouse with an advanced tumor and the other with an early tumor, resulted in a pronounced inhibition of growth of the early tumor. Based upon these observations, [Bichel, Europ. J. Cancer 6: 291-296 (1970) and Bichel, supra] the existence of a diffusible inhibitory principle which circulated through the peritoneum of parabiotic mice and was present in the cell-free ascites fluid produced by the fully developed ascites tumors was postulated. The nature of this inhibiting principle was not characterized, but it was speculated that the rate of growth of ascites tumors was dependent upon the amount of tumor tissue present in the mouse and that the amount of tumor tissue was determined by the amount inhibitory principle produced.
Substances having tumor growth inhibitory activity have been described. McMahon, et al. [Proc. Natl. Acad. Sci. USA 79, 456-460 (1982)] have purified from rat liver a 26,000 dalton substance which inhibits the proliferation of nonmalignant rat liver cells, but does not inhibit the proliferation of malignant rat liver cells. Other growth inhibitory substances have been identified in cultured chicken spinal cord cells [Kage, et al., Experimental Neurology 58: 347-360 (1970); Harrington, et al., Proc. Natl. Acad. Sci. USA 77: 423-427 (1980) and Steck, et al., J. Cell Biol. 83: 562-575 (1979)].
Holley et al., [Proc. Natl. Acad. Sci. 77; 5989 (1980) and Cell Biol. Int. Reports 7: 525-526 (1983)] reported that a substance isolated from African green monkey BSC-1 cells inhibited the growth of BSC-1 cells, human mammary tumor cells and normal human mammary cells. More recently, biochemical characterization of this inhibitory substance [Tucker, et al., Science 226: 705-707 (1984); Roberts, et al. Proc. Nat. Acad. Sci. 82: 119-123 (1985)] showed it to be identical, or highly related, to a 25,000 dalton two chain human platelet-derived polypeptide designated TGF-xcex2 [Assoian, et al., J. Biol. Chem 258: 7155-7160 (1983)]. TGF-xcex2 derived from either human platelets [Sporn and Roberts, international patent number WO 84/01106] or from human placenta [Frolik et al., (1983) PNAS 80 3676-3680; Sporn and Roberts (WO84/01106)] induces anchorage independent colony growth in soft agar of non-neoplastic rat kidney fibroblasts and other cells in the presence of transforming growth factor alpha or epidermal growth factor. More recently, the bifunctional nature of this molecule as a regulator of cellular growth has been confirmed by Roberts et al. [Proc. Natl. Acad. Sci. 82: 119-123 (1985)]. Iwata et al., [J. Cellular Biochem. Suppl. 5: 401 (1982)] previously described a microtiter plate system for assaying growth stimulation and growth inhibition activity. Todaro et al., [Todaro et al., in Tumor Cell Heterogeneity; Origins and Implications, Bristol-Myers Cancer Symposia, Volume 4, Owens, A. H., Coffey, D. S., and Baylin, S. B., Eds. (Academic Press, 1982), pp. 205-224)] and Iwata et al., [Fed. Proc. Fed. AM. Soc. Exp. Biol. 42: 1833 (1983)] reported the isolation of tumor inhibitory activity from tissue culture fluids of human tumor cells propagated in culture. The observations described in these reports were preliminary and little detail was provided.
On Apr. 20, 1984, a patent application was filed with the United States Patent and Trademark Office under U.S. Ser. No. 602,520, entitled xe2x80x9cSubstantially Purified Tumor Growth Inhibitory Factor (TIF)xe2x80x9d on which one of us, Kenenth K. Iwata, is named as coinventor. This application concerns the preliminary identification of a not well-defined substance or substances present in, and derived from, human tumor cells propagated in culture. This substance or substances resembles the tumor inhibitory activity previously reported. [Todaro, et al., in Tumor Cell Heterogeneity; Origins and Implications, Bristol-Myers Cancer Symposia, Volume 4, Owens, A. H., D. S., and Baylin, S. B., Eds. (Academic Press, 1982), pp. 205-224; Iwata, et al., Fed. Proc. Fed. Am. Soc. Exp. Biol. 42: 1833 (1983).]
Todaro [Todaro, G. J. in Epigenetic Regulation of Cancer, Terry Fox Cancer Research Conference (University of British Columbia; Vancouver, B.C., Canada) Abs. 13 (1984)] subsequently reported two factors with tumor cell growth inhibitory properties which were reportedly sequenced and shown to consist of 70 and 90 amino acid residues, respectively. However, Todaro failed to report the source of the factors, their tissue type, the species the factors were derived from or the method of the factor purification.
The invention concerns an acidified, ethanol extract derived from human tissue which comprises a plurality of acidic proteins, each of which has a molecular weight of about 26,000 daltons, is a dimer composed of two polypeptides each of which has an apparent molecular weight of about 13,000 daltons and is joined to the other one by one or more disulfide bonds. This extract has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts. The inhibitory activity against human tumor cell growth is not destroyed upon increasing the temperature of the acidified, ethanol extract to about 100xc2x0 C. for about 3 minutes or upon adding acetic acid and the inhibitory activity is enhanced when the acidified, ethanol extract is prepared at about 4xc2x0 C. rather than about 23xc2x0 C.
This invention also concerns an acidified, ethanol extract derived from human umbilical cord which has been treated to remove substantially all blood, all extracellular soluble components and substantially all intracellular soluble components, which comprises at least two acidic proteins each of which has an apparent molecular weight of about 26,000 daltons and each of which is a dimer composed of two polypeptides having apparent molecular weights of about 13,000 daltons and being joined to each other by disulfide bonds under nonreducing conditions. Each extract has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts, the inhibitory activity against human tumor cell growth not being destroyed upon increasing the temperature of the acidified, ethanol extract to about 100xc2x0 C. for about 3 minutes or upon adding acetic acid until the acidified, ethanol extract is up to about 1.0 molar in acetic acid.
The invention further concerns a method for preparing an acidified, ethanol extract from human tissue, e.g., human umbilical cord or human placenta, the acidified, ethanol extract comprising a plurality of acidic proteins, each of which has a molecular weight of about 26,000 daltons and each of which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibro-blasts. The method comprises treating under suitable conditions, the tissue source to produce acid soluble proteins, recovering the solubilized proteins, separately recovering from the solubilized extract proteins having an apparent molecular weight of about 26,000 daltons, assaying the proteins so recovered to identify those which either inhibit the growth of human tumor cells or inhibit the growth of an established mink lung cell line (CCL 64), or enhance the growth of normal human foreskin fibroblasts and recovering an acidified, ethanol extract containing the proteins so identified.
This invention also concerns a method for preparing an acidified, ethanol extract from human umbilical cord, the acidified, ethanol extract comprising tissue-derived growth inhibitor (TGI), a mixture of proteins of apparent molecular weight of about 26,000 daltons, which has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) while stimulating the growth of normal human foreskin fibroblasts. The method comprises under suitable conditions, removing the tissue, washing the tissue to remove all traces of blood and contaminating proteins, solubilizing and isolating the remaining proteins from the tissue by acidified ethanol extraction to produce solubilized proteins, separately recovering from the solubilized TGI proteins having an apparent molecular weight of about 26,000 daltons, assaying the separately recovered TGI proteins to identify the activity which inhibits the growth of human tumor cells, inhibits the growth of an established mink lung cell line (CCL 64) and enhances the growth of normal human foreskin fibroblasts, and recovering the acidified, ethanol extract containing the TGI so identified.
The invention additionally provides a composition of matter designated tissue-derived growth inhibitor-1 (TGI-1) which comprises a protein which is a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds. TGI-I has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts. TGI-I has an apparent molecular weight of about 26,000 daltons, and is recoverable as a defined activity on hydrophobic interaction chromatography on phenyl-Sepharose after ether-ethanol precipitation at about 1.5M ammonium acetate and 31% ethylene glycol and on high performance liquid chromatography of an acidified, ethanol extract with a separating gradient of acetonitrile on a xcexcBondapack(copyright) C18 column containing 0.05% trifluoracetic acid at about 27% acetonitrile and recoverable as a defined activity on high performance liquid chromatography of an acidified, ethanol extract with a separating gradient of 2-propanol containing 0.05% trifluoracetic acid at about 40-41% 2-propanol. The invention additionally provides a method for preparing TGI-1.
The invention also provides a composition of matter designated tissue-derived growth inhibitor (TGI) which comprises at least two proteins and has the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts, each protein having an apparent molecular weight of about 26,000 daltons. Their composition is recoverable as a defined activity on high performance liquid chromatography of an acidified, ethanol extract with a linear gradient of acetonitrile containing 0.05% trifluoracetic acid at about 28-34% acetonitrile and is resolved as a single peak of defined activity from a cation exchange resin when eluted by a linear NaCl gradient at about 0.6-0.7 M NaCl and is recoverable as a defined activity on high performance liquid chromatography of an acidified, ethanol extract from human umbilical cord with a separating gradient of phenyl-Sepharose.
The invention further provides a composition of matter designated tissue-derived growth inhibitor-2 (TGI-2) which comprises a protein having the property of inhibiting the growth of human tumor cells and of an established mink lung cell line (CCL 64) but not the growth of normal human foreskin fibroblasts and having an apparent molecular weight of about 26,000 daltons. TGI-2 is a dimer composed of two polypeptides each having an apparent molecular weight of about 13,000 daltons and being joined to each other by disulfide bonds. TGI-2 is recoverable as a defined activity on hydrophobic interaction chromatography on a phenyl-Sepharose after ether-ethanol precipitation at about 1.5M ammonium acetate and 31% ethylene glycol and on high performance liquid chromatography of an acidified, ethanol extract with a separating gradient of acetonitrile containing 0.05% trifluorocetic acid at about 28-30% acetonitrile on a C18 column and is recoverable as a defined activity on high performance liquid chromatography of an acidified, ethanol extract with a linear gradient of 2-propanol on a CN column containing 0.05% trifluoracetic acid at about 44% 2-propanol. The invention additionally provides a method for preparing TGI-2.
The invention also provides a polypeptide from conditioned media of A431 cells having an apparent molecular weight less than about 30,000 daltons and having the property of substantially inhibiting the growth of a human tumor cell line (A549) but not of an established mink lung cell line (CCL 64).
Furthermore, the invention provides a polypeptide designated CM-I which has the property of substantially inhibiting the growth of human tumor cells but not the growth of an established mink lung cell line (CCL 64).
This invention also provides a protein having an apparent molecular weight of about 26,000 daltons. The protein is a dimer composed of two polypeptides having an apparent molecular weight of about 13,000 daltons and being joined by disulfide bonds. The protein demonstrates tumor growth inhibitory activity of human tumor cell line (A549) and of an established mink lung cell line (CCL 64). It is acid soluble in 1.0 M acetic acid; and 0.1% trifluoracetic acid and is stable in heat to about 100xc2x0 C.; and is stable in up to 39% acetonitrile and 45% 2-propanol. The protein may be designated TGI-1 or TGI-2.
This invention also provides a protein having tumor growth inhibitory activity comprising the 112 amino acids shown in FIG. 29 beginning with alanine at position 1 and ending with serine at position 112. Preferably, this protein is a purified protein having 112 amino acids beginning with alanine at position 1 and ending with serine at position 112 as shown in FIG. 29. The protein may also comprise the 205 amino acids shown in FIG. 29 beginning with serine at position xe2x88x9293 and ending with serine at position 112. Thus, this 205 amino acid sequence contains partial precursor sequence of the protein having tumor growth inhibitory activity with the complete sequence of the mature protein.
This invention further provides a nucleic acid molecule encoding the protein having tumor growth inhibitory activity comprising the 112 amino acids shown in FIG. 29 beginning with alanine at position 1 and ending with serine at position 112. The nucleic acid molecule may encode the entire protein shown in FIG. 29 beginning with serine at position xe2x88x9293 and ending with serine at position 112. Alternatively, the nucleic acid molecule may encode only the 112 amino acids found in the functional protein shown in FIG. 29 beginning with alanine at position 1 and ending with serine at position 112. These nucleic acid molecules may be cDNA, genomic DNA, or mRNA.
This invention also provides a plasmid which comprises the nucleic acid molecules of this invention as well as a host vector system comprising the plasmid in a suitable host cell. As one embodiment of this invention, a pUC8 plasmid containing a 1.7 kb EcoRI insert was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va., 20110-2209, U.S.A., pursuant to the Budapest Treaty on Dec. 20, 1990 and was accorded ATCC Designation No. 40939. This host vector system comprises any plasmid and vector known in the art which are suitable for producing the proteins of this invention. The suitable host cell may be a bacteria cell or a eucaryotic cell.
This invention further provides a method for producing a protein comprising growing the host vector system of this invention so as to produce the protein in the host and recovering the protein so produced.
Pharmaceutical compositions which comprise effective amounts of one of the extracts, TGI-1, TGI, TGI-2, the protein having tumor growth inhibitory activity, the polypeptide recoverable from conditioned media of A431 cells, or CM-I, and a suitable pharmaceutical carrier are provided, as are methods for inhibiting the growth of human tumor cells by contacting the cells with such compositions. The compositions may also be used to treat burns or in wound healing.
The invention also concerns methods for detecting the presence of tumors which comprise quantitatively determining the amount of TGI-1, TGI or TGI-2, the protein having tumor growth inhibitory activity, the polypeptide recoverable from conditioned media of A431 cells, or CM-I present in a sample from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a significantly different amount indicating the presence of a tumor. Further, the invention concerns methods for detecting the presence of tumors which comprise separately quantitatively determining the amount of TGI-1, TGI, TGI-2, the protein having tumor growth inhibitory activity, CM-1 or the polypeptide recoverable from conditioned media of A431 cells, and the amount of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of TGI-1, TGI, TGI-2, the protein having tumor growth inhibitory activity, CM-I or the polypeptide recoverable from conditioned media of A431 cells, or of a heterogeneous mixture thereof, present in the sample to the amount of TGF-alpha present, determining the comparable ratio for a sample from a normal subject and comparing the ratio for the subject with the ratio for the normal subject, a significant variation in these ratios indicating the presence of a tumor.
Finally, the invention concerns a method for typing tumors which comprises determining from a sample of a subject with a tumor the presence of one or more of TGI-1, TGI, TGI-2, the protein having tumor growth inhibitory activity, CM-I, or the polypeptide recoverable from condition media of A431 cells in the sample, the presence or absence of a specific combination thereof, or the presence of specific amounts or relative amounts thereof, being indicative of a specific tumor type.